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. 2014 Mar;24(3):331-43.
doi: 10.1038/cr.2014.10. Epub 2014 Jan 24.

VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex

Wenjing Zhang  1 , Yijun Gao  1 , Peixue Li  1 , Zhubing Shi  1 , Tong Guo  1 , Fei Li  1 , Xiangkun Han  1 , Yan Feng  1 , Chao Zheng  1 , Zuoyun Wang  1 , Fuming Li  1 , Haiquan Chen  2 , Zhaocai Zhou  1 , Lei Zhang  1 , Hongbin Ji  1
Affiliations

VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex

Wenjing Zhang et al. Cell Res. 2014 Mar.

Erratum in

Abstract

Lung cancer is one of the most devastating diseases worldwide with high incidence and mortality. Hippo (Hpo) pathway is a conserved regulator of organ size in both Drosophila and mammals. Emerging evidence has suggested the significance of Hpo pathway in cancer development. In this study, we identify VGLL4 as a novel tumor suppressor in lung carcinogenesis through negatively regulating the formation of YAP-TEAD complex, the core component of Hpo pathway. Our data show that VGLL4 is frequently observed to be lowly expressed in both mouse and human lung cancer specimens. Ectopic expression of VGLL4 significantly suppresses the growth of lung cancer cells in vitro. More importantly, VGLL4 significantly inhibits lung cancer progression in de novo mouse model. We further find that VGLL4 inhibits the activity of the YAP-TEAD transcriptional complex. Our data show that VGLL4 directly competes with YAP in binding to TEADs and executes its growth-inhibitory function through two TDU domains. Collectively, our study demonstrates that VGLL4 is a novel tumor suppressor for lung cancer through negatively regulating the YAP-TEAD complex formation and thus the Hpo pathway.

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Figures

Figure 1
Figure 1
VGLL4 is lowly expressed in both human and mouse lung adenocarcinomas. (A) Real-time PCR quantification of Vgll4 mRNA levels in mouse KrasG12D lung adenocarcinoma and normal lungs. Data are shown as means ± SEM. *P < 0.05. (B) H&E and Vgll4 immunohistochemical staining on mouse KrasG12D lung adenocarcinoma and normal lungs. Scale bar, 50 μm. (C) Quantification of VGLL4 mRNA levels in human lung adenocarcinoma and paired pathologically normal lungs by real-time PCR. The values were presented as log10 ratio of the VGLL4 expression of human lung adenocarcinomas vs normal lung specimens. (D) Immunohistochemical staining of VGLL4 on human lung adenocarcinoma and normal lungs. Scale bar, 500 μm (top) and 50 μm (bottom). (E) Statistical analysis of nuclear VGLL4 staining in human lung adenocarcinoma and normal lung specimens. ADC, adenocarcinoma; NL, normal lung.
Figure 2
Figure 2
VGLL4 inhibits lung cancer cell growth in vitro. (A and B) The proliferations of A549 (A) and CRL-5872 (B) cells with or without ectopic VGLL4 expression were detected by MTT assay. Data are shown as means ± SEM. ***P < 0.005. (C and D) Cell cycle distributions of A549 (C) and CRL-5872 (D) cells with or without VGLL4 overexpression were assessed by FACS assay. (E and F) Colony formation abilities of A549 (E) and CRL-5872 (F) cells with or without ectopic VGLL4 expression were assessed by soft agar assay. Data are shown as means ± SEM. **P < 0.01; ***P < 0.005. (G and H) A549 (G) and CRL-5872 (H) cells with or without VGLL4 overexpression were cultured in Matrigel for 7 days. Colony size was shown by photos. Scale bar, 0.01 mm.
Figure 3
Figure 3
VGLL4 significantly suppresses lung cancer progression in de novo mouse model. (A) Scheme of viral infection strategy for ectopic VGLL4 expression in Lkb1 L/L/KrasG12D mouse model. (B) VGLL4 immunohistochemical staining in lung tumors of Lkb1L/L/KrasG12D mouse at 13 weeks post viral infection with Lenti-Cre or Lenti-VGLL4-Cre. Scale bar, 50 μm. (C) Representative H&E photos of Lkb1L/L/KrasG12D mice lung at 13 weeks post viral infection with Lenti-Ctrl-Cre or Lenti-VGLL4-Cre. Scale bar, 500 μm (top) and 50 μm (bottom). (D) Statistical analysis of lung tumors with the indicated size from Lkb1L/L/KrasG12D mice virally infected with Lenti-Ctrl-Cre or Lenti-VGLL4-Cre. Data are shown as means ± SEM. *P < 0.05. (E) Ki-67 immunohistochemical staining of lung sections from Lkb1L/L/KrasG12D mice virally infected with Lenti-Ctrl-Cre or Lenti-VGLL4-Cre. Scale bar, 50 μm. (F) Quantitative analyses of proliferative index in lung tumors from Lkb1L/L/KrasG12D mice virally infected with Lenti-Ctrl-Cre or Lenti-VGLL4-Cre. Data are shown as means ± SEM. *P< 0.05.
Figure 4
Figure 4
TEADs mediate VGLL4's inhibitory role in lung tumor cell growth. (A) Co-immunoprecipitation experiments of TEAD4 with wild-type (WT) VGLL4 or VGLL4 mutant with deletion of two TDU domains in HEK-293T cells. TEAD4 is HA-tagged and VGLL4 is FLAG-tagged. (B and C) Deletion of two TDU domains abolishes the inhibitory role of VGLL4 in the cell proliferation of human lung cancer cell lines A549 (B) and CRL-5872 (C). Data are shown as means ± SEM. **P < 0.005. (D and E) Deletion of two TDU domains abolishes the inhibitory role of VGLL4 in the colony formation of human lung cancer cell lines A549 (D) and CRL-5872 (E) in soft agar. *P< 0.05; **P< 0.01.
Figure 5
Figure 5
VGLL4 inhibits the transcriptional activity of TEADs and the expression of their downstream target genes. (A) Ectopic expression of wild-type (WT) VGLL4 but not VGLL4 mutant with deletion of two TDU domains decreases TEAD4-dependent luciferase reporter activity in HEK-293T cells. Data are shown as means ± SEM. ***P< 0.005. (B and C) Ectopic expression of WT VGLL4 but not VGLL4 mutant with deletion of two TDU domains significantly downregulates the expression of TEAD downstream target genes (CTGF and CYR61) in HEK-293T cells (B) and A549 cells (C). Data are shown as means ± SEM. *P< 0.05; **P < 0.01; ***P< 0.005. (D) Knockdown of VGLL4 increases TEAD4-dependent luciferase reporter activity in HEK-293T cells. Data are shown as means ± SEM. **P < 0.01; ***P < 0.005. (E and F) Knockdown of VGLL4 upregulates the transcripts of CTGF and CYR61 in Beas2B (E) and CRL-5807 (F) cells. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005.
Figure 6
Figure 6
VGLL4 inhibits the growth-promotive function of YAP through the direct competition for its binding to TEAD4. (A) Ectopic VGLL4 expression decreases the TEAD4-dependent luciferase reporter activity promoted by YAP overexpression in HEK-293T cells. Data are shown as means ± SEM. ***P < 0.005. (B and C) Ectopic VGLL4 expression decreases YAP-induced CTGF and CYR61 expression in HEK-293T cells (B) and A549 cells (C). Data are shown as means ± SEM. **P < 0.01; ***P < 0.005. (D) VGLL4 directly competes with YAP for binding to TEAD4 in vitro. YAP, amino acids 50-504 containing TEAD-binding domain. TEAD4, amino acids 217-434; VGLL4, amino acids 206-259 containing two TDU domains. (E) Ectopic VGLL4 expression significantly suppresses the growth-promotive function of YAP in A549 cells. Data are shown as means ± SEM. ***P < 0.005. (F) Ectopic expression of VGLL4 significantly inhibits the colony formation of A549 cells promoted by YAP overexpression. *P < 0.05.
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